Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Back-transfer of late gastrula nuclei of nucleocytoplasmic hybrids

Late gastrula-early neurula endoderm nuclei from nucleocytoplasmic hybrids consisting of Rana pipiens nuclei and Rana palustris cytoplasm were back-transferred into enucleated eggs of Rana pipiens. Approximately half of the normally cleaved back-transfer embryos developed normally. A similar result was obtained with nuclear transfer controls. As expected, the development of the nucleocytoplasmic hybrid donors, at the time the back-transfers were carried out, was clearly abnormal. Consequently, a significant proportion of endoderm nuclei from these embryos undergo no irreversible changes in their developmental capacity even after the nucleocytoplasmic incompatibility between these two amphibian species is expressed.     

Meiotic maternal chromosomes introduced to the late mouse zygote are recruited to later embryonic divisions

The aim of this study was to investigate the fate of an additional female genome introduced to a dividing zygote. Maternal chromatin in the form of karyoplasts containing a metaphase II spindle were fused to zygotes blocked in anaphase or telophase of the first cleavage. Permanent preparations made 20-40 min after fusion at anaphase revealed that the donor maternal chromosomes had entered anaphase or telophase in 16 out of 18 cases. A further two groups of embryos that were fused at either anaphase or anaphase/telophase were cultured to the first division. Division occurred 50 min after fusion in both groups of embryos (86 and 85.1%, respectively), of which most divided to two cells (80 and 71.6% of total) and the remainder divided to three cells. About two thirds of two-cell embryos contained an extra nucleus in one blastomere. Nuclei containing donor maternal chromosomes reached a similar size to recipient nuclei in 68% of embryos derived from anaphase-blocked zygotes, in contrast to 31.1% of embryos derived from anaphase/telophase-blocked embryos. Replication of DNA in donor nuclei closely followed the timing and intensity of that in control embryos. When fixed 24 hr after fusion, one third of embryos were still at the two-cell stage, with one or both blastomeres showing a single metaphase plate of the second cleavage. In the remaining embryos, three or four cells were present, some containing two nuclei. Blastocysts developed in 50% of fused embryos and three young were born after transfer of cleaving hybrid embryos to recipients. Chromosome preparations from bone marrow of the young contained 3-4 tetraploid metaphase plates per several hundred plates counted compared with none in control embryos. In conclusion, additional maternal chromosomes can be introduced at the late-dividing zygote and join the embryonic cell cycles during subsequent divisions. This method may provide a useful approach for studying changes specific to the maternal genome during early cell cycles of the mammalian embryo.     

Control of first cleavage in single-cell reconstituted mouse embryos

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.     

Nuclear transplantation in mouse embryos: assessment of recipient cell stage

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos. Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes. Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation. Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos. No development was observed beyond Day 12 of gestation. These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote.     

Nuclear transfer of mouse follicular epithelial cells pretreated with spermine, protamine, or putrescine

The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 microg/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. J. Exp. Zool. 289:208-212, 2001.     

Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.     

Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.     

Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes

The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbecco's modified Eagle's medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.     

Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal and adult somatic cell nuclei into bovine (Bos indicus) oocyte cytoplasm and their subsequent development

Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal fibroblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal fibroblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal fibroblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a significantly higher cleavage rate (P < 0.05) for fused nuclear transferred embryos produced by fetal fibroblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal fibroblasts was higher (P < 0.05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts confirmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells.     

硬骨鱼类体细胞核移植的研究

本文用不同属、科、目的硬骨鱼类作材料进行体细胞核移植研究。鲫鱼（Ｃａｒａｓｓｉｕｓａｕｒａｔｕｓ）、鲮鱼（Ｃｉｒｒｈｉｎｕｓｍｏｌｉｔｏｒｅｌｌａ）和尼罗罗非鲫（Ｔｉｌａｐｉａｎｉｌｏｔｉｃａ）的体细胞核（头肾细胞）移植到鲤鱼（Ｃｙｐｒｉｎｕｓｃａｒｐｉｏ）的成熟去核卵中,通过继代核移植,在鲫鱼体细胞核和鲤鱼去核卵的属间组合中,获得发育到血液循环期的幼鱼;在鲮鱼体细胞核和鲤鱼去核卵的亚科间组合中,获得发育到心脏跳动期的晚期胚胎;在尼罗非鲫体细胞核和鲤鱼去枚卵的目间组合中,获得发育到肌肉效应期的胚胎。由于是直接用成鱼体细胞核作供核体进行核移植,因而能够克服供体鱼和受体鱼不同步产卵的困难。实验结果表明,这对进行硬骨鱼类核质杂交研究无疑是一种简便而又有效的方法。     

Die Zytologie der bisexuellen und parthenogenetischen Fortpflanzung von Heteropeza pygmaea Winnertz,einer Gallmücke mit pädogenetischer Vermehrung

Heteropeza pygmaea (syn. Oligarces paradoxus) can reproduce as larvae by paedogenesis or as imagines (Fig. 1). The eggs of imagines may develop after fertilization or parthenogenetically. The fertilized eggs give rise to female larvae, which develop into mother-larvae with female offspring (Weibchenmütter). Only a few of the larvae which hatch from unfertilized eggs become motherlarvae with female offspring; the others die. Spermatogenesis is aberrant, as it is in all gall midges studied to date. The primary spermatocyte contains 53 or 63 chromosomes. The meiotic divisions give rise to two sperms each of which contains only 7 chromosomes (Figs. 5–11). The eggs of the imago are composed of the oocyte and the nurse-cell chamber. In addition to the oocyte nucleus and the nurse-cell nuclei there are three other nuclei in the eggs (Figs. 15–17). They are called small nuclei (kleine Kerne). In prometaphase stages of the first cleavage division it could be seen that these nuclei contain about 10 chromosomes. Therefore it is assumed that these nuclei originate from the soma of the mother-larva. The chromosome number of the primary oocyte is approximately 66. The oocyte completes two meiotic divisions. The reduced egg nucleus contains approximately 33 chromosomes. The polar body-nuclei degenerate during the first cleavage divisions. The fertilized egg contains 2–3 sperms. The primary cleavage nucleus is formed by the egg nucleus and usually all of the sperm nuclei and the small nuclei (Figs. 21–29). The most frequent chromosome numbers in the primary cleavage nuclei are about 77 and 67. The first and the second cleavage divisions are normal. A first elimination occurs in the 3rd, 4th, and 5th cleavage division (Fig. 30). All except 6 chromosomes are eliminated from the future somatic nuclei. Following a second elimination (Figs. 33, 34), the future somatic nuclei contain 5 chromosomes. No elimination occurs in the divisions of the germ line nucleus. In eggs which develop parthenogenetically the primary cleavage nucleus is formed by the egg nucleus and 2–3 small nuclei. It's chromosome number is therefore about 53 or 63. After two eliminations, which are similar to the ones which occur in fertilized eggs, the soma contains 5 chromosomes. The somatic nuclei of male larvae which arrise by paedogenesis contain 5 chromosomes; while the somatic nuclei of female larvae of paedogenetic origin contain 10 chromosomes. It was therefore assumed earlier that sex was determined by haploidy or diploidy. But the above results show that larvae from fertilized as well as from unfertilized eggs of imagines have 5 chromosomes in the soma, but are females, and the female paedogenetic offspring of larvae from unfertilized eggs have either 5 or 10 chromosomes in their somatic cells. Therefore sex determination is not by haploidy-diploidy but by some other, unknown, mechanism. The cytological events associated with paedogenetic, bisexual, and parthenogenetic reproduction in Heteropeza pygmaea are compared (Fig. 37). The occurrence and meaning of the small nuclei which are found in the eggs of most gall midges are discussed. It has been shown here that these nuclei function to restore the chromosome number in fertilized eggs; it is suggested that they function similarity in certain other gall midges. Consideration of the mode of restoration of the germ-line chromosome number leads to the conclusion that in Heteropeza few, if any, of the chromosomes are limited to the germ-line, i.e. can never occur in somatic cells (p. 124).     

Light microscopical studies of chromosomes in embryos of cod, Gadus morhua L.

The chromosomes in the cells of 1–2-day-old cod embryos were studied using light microscopic techniques. The observations revealed slightly non-synchronous cell divisions, with at least three different mitotic phases present in a 1-day-old embryo. Surprisingly high percentages of abnormal metaphases and anaphases were found in normal-looking embryos. In the 2-day-old embryos, free-lying nuclei were observed in the periblast zone. Chromosome studies of these nuclei revealed a cleavage pattern of the chromosomes which differed from that of the other cells.     

The cytology of paedogenesis in the gall midgeMycophila speyeri

In paedogenetically developing female eggs of the gall midgeMycophila speyeri only one equational meiotic division occurs. The primary cleavage nucleus contains 29 chromosomes. In the fourth cleavage division 23 chromosomes are eliminated from the future somatic nuclei while the primordial germ-line nucleus keeps the high chromosome number.—The paedogenetic development of male eggs begins with two meiotic divisions. The egg nucleus with 14 or 15 chromosomes fuses with two, sometimes only one, somatic nuclei (2n=6) of maternal origin (regulation). Thus the primary cleavage nucleus contains 26 or 27 chromosomes, sometimes only 20 or 21. Elimination in cleavage divisions V and VI leeds to somatic nuclei with 3 chromosomes while the primordial germ-line nucleus keeps the high chromosome number.—Differences between male and female eggs and the evolution of regulation in gall midges are discussed.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Nuclear transplantation in early pig embryos

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) × Orychophragmus violaceus hybrids

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n=54, AABBCC) and another crucifer Orychophragmus violaceus (2n=24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.     

Chromosomal transplantation

Dissociated cells of middle-to-late blastulae were exposed to 0.1 mg colchicine/ml and achieved 92% metaphase arrest. These cells contained a haploid set of Bombina maxima (Anura:Discoglossidae) chromosomes. When transplanted into the enucleated eggs of B. orientalis, some donor cells stimulated development to the late blastula and middle gastrula stages. — Most (17/20) of the embryos resulting from chromosomal transplantation were nonmosaic aneuploids. A high percentage of recipient egg enucleation (93%), the ratio of long-to-short chromosomes, and the presence of species-specific marker chromosomes proved that chromosomes were transplanted from the donor cells. Therefore, metaphase chromosomes lacking intact spindle apparatuses were injected into and incorporated by amphibian eggs. These chromosomes were replicated in all cells of the resulting embryos. The aneuploidy of these embryos is explained by an inability of the recipient egg to locate and replicate many transplanted chromosomes (44%) before first cleavage.